Pre-conditioning cells against death

ABSTRACT

Apoptosis and/or necrosis related disorders in the mammalian body, namely radiation exposure disorders, chemical exposure and ingestion disorders, neurological disorders and physical trauma disorders, are treated, and their onset is counteracted by preconditioning, by extracting from the mammalian body an aliquot of blood, subjecting the extracted aliquot, ex vivo, to an oxidative stressor such as exposure to ozone gas, a temperature stressor, i.e. temperatures above or below body temperatures, and ultraviolet light, and re-injecting the treated blood aliquot into the mammalian body. The treatment ha the effect of decreasing apoptosis/necrosis in the body, and of pre-conditioning the body better to withstand subsequently encountered apoptosis-inducing events.

FIELD OF THE INVENTION

This invention relates to the field of medicine. More specifically, theinvention relates to means for preconditioning the mammalian body,including the human body, so as to enable cellular organs thereof betterto resist subsequently encountered cell death as induced byapoptosis-inducing events, or by necrosis-inducing events, includingevents inducing both apoptosis and necrosis.

BACKGROUND OF THE INVENTION

The two known forms of cellular death are necrosis and apoptosis.Apoptosis is the biological process of controlled, programmed celldeath, by means of which cells die by a process of condensation withoutthe release of cell contents into the surrounding milieu. Cells of mostorgans and tissues divide and multiply over time, a process that isnormally in equilibrium with cell death by apoptosis, resulting inoptimal cell numbers in the healthy body. Apoptosis, therefore, can beconsidered to act as a control on the total number of cells in organsand tissues. Residues of apoptosed cells are largely consumed by othercells, by a process of phagocytosis. The process of apoptosis, thenatural, well-regulated process by which the body undertakes removal ofunwanted cells is to be contrasted with the process of necrosis by whichthe cells die largely in an uncontrolled manner, as a result of membranerupture. Importantly, however, in many instances apoptosis and necrosisbehave as a continuum. The intracellular components of necrosed cellsare released into the organism in an uncontrolled manner, commonlyresulting in inflammatory reactions as the body attempts to deal withthese suddenly encountered components. Apoptosed cells cause virtuallyno harmful inflammatory reactions.

Some medical disorders in a living body, or an individual organ of aliving body, can be attributed at least in part to an undue accelerationin the rate of apoptosis. This can occur, for example, when a bodyingests chemical poisons or encounters excessive amounts of harmfulradiation (radioactivity, UV exposure, etc.). Other disorders involveboth apoptosis and necrosis. Still other disorders involve anaccelerated rate of cell death due primarily to necrosis.

Apoptosis of the cell is understood to be initiated by an alteration inthe functioning of the mitochondria of the cell. Mitochondria, as iswell known, are membrane-bounded organelles, located within the cell,and occupying a major fraction of the total cell volume. They containlarge amounts of internal membrane. The main function of mitochondria isto convert energy from foodstuffs to forms that can be used to drivecellular reactions. This is accomplished by a process of chemiosmoticcoupling, by which membrane-bound ion pumps transfer ions from one sideof the mitochondrial membrane to the other. The proton pumps generate anelectrochemical proton gradient across the membrane, which is used todrive various energy-requiring reactions when the protons flow throughmembrane embedded proteins such as the enzyme ATP synthase. As an ionicprocess, the potential across the mitochondrial membrane is important inthe efficient operation of this energy-providing mechanism. Mitochondriaparticipate directly in the induction of apoptosis by releasingpro-apoptotic proteins Decreases in mitochondrial membrane potential areknown to be indicative of the commencement of apoptosis.

Organs undergoing apoptosis exhibit oligonucleosomal DNA fragmentationinto 180-200 base pairs, in a specific pattern which appears as a ladderafter gel electrophoresis. The degree of DNA fragmentation correlateswith the progression of apoptosis in the organ, and can be measured byextracting the DNA, radiolabelling it, subjecting it to electrophoresisand quantifying the radioactivity associated with various DNA fragments.Such techniques can be used to determine the numbers of cells undergoingapoptosis or exhibiting an apoptotic condition or predisposition, so asto determine an extent or degree of apoptosis in a body organ or tissue.

In the course of necrosis, enzymes and other cell contents normallycontained in the cytoplasm are released, as a result of disintegrationof cell membranes, a hallmark of necrosis. One of these is the enzymelactate dehydrogenase (LDH), the levels of which are commonly used todetermine the degree of necrosis.

BRIEF REFERENCE TO THE PRIOR ART

U.S. Pat. No. 4,968,483 Mueller et al. describes an apparatus foroxygenating blood, by treating an aliquot of a patient's bloodextracorporeally, with an oxygen/ozone mixture and ultraviolet light, ata controlled temperature. The apparatus is proposed for use inhematological oxidation therapy.

U.S. Pat. No. 5,591,457 Bolton, discloses a method of inhibiting theaggregation of blood platelets in a human, a method of stimulating theimmune system and a method of treating peripheral vascular diseases suchas Raynaud's disease, by extracting an aliquot of blood from a patient,subjecting it to ozone/oxygen gas mixture, and ultraviolet radiation ata temperature in the range of about 37-43° C., and then reinjecting thetreated blood in the human patient.

U.S. Pat. No. 5,834,030 Bolton, describes a similar process forincreasing the content of nitric oxide in the blood of a mammalianpatient, potentially useful in treating conditions such as high bloodpressure in mammalian patients.

International Patent Application PCT/CA97/00564 Vasogen Inc.(WO98/07436) described an autoimmune vaccine for administration to humanpatients to alleviate the symptoms of autoimmune diseases such asrheumatoid arthritis, the vaccine comprises an aliquot of the patient'sblood which has been subjected extracorporeally to an oxidizingenvironment, UV radiation and elevated temperature.

It is an object of the present invention to provide means forpre-conditioning a mammalian patient to better withstand externalcellular insults likely to effect acceleration of or to increase thedegree of apoptosis in tissues or organs of the mammalian patient.

It is a further object of the invention to provide means forpre-conditioning a mammalian patient to better withstand externalcellular insults likely to effect acceleration of or increase the degreeof necrosis in tissues or organs of the patient.

It is a further object of the invention to provide a pre-conditioningprocess for mammalian patients against the harmful effects of chemicaland radiation poisoning.

It is a further object of the invention to provide a process foralleviating or decelerating the progression of the symptoms ofapoptosis-related or necrosis-related medical disorders.

SUMMARY OF THE INVENTION

The present invention provides a process whereby a mammalian body may bepreconditioned so that the cells of organs and tissues can better resistsubsequently encountered apoptosis- and/or necrosis-inducing events. Theprocess involves in vitro treatment of an aliquot of the blood from themammalian body, with certain stressors to effect modification of theblood aliquot. Then the treated blood aliquot is reintroduced into themammalian body. The result is a significant increase in resistance toapoptosis and apoptosis/necrosis of the cells of the body, as indicatedby changes in mitochrondrial membrane potential, decrease of DNAladdering, and decrease of release of LDH, when the cells aresubsequently exposed to stressing or toxic agents.

The aliquot of blood is treated by being subjected to one or morestressors which have been found to modify the blood. According to thepresent invention, the blood aliquot can be modified by subjecting theblood, or separated cellular or non-cellular fractions of the blood, ormixtures of the separated cells and/or non-cellular fractions of theblood, to stressors selected from heat, ultraviolet light and oxidizingenvironments. The stressors may be applied individually, or in anycombination of two or more of such stressors, simultaneously orsequentially.

Accordingly, the process of the invention may be used forpre-conditioning the mammalian body against the effects of a wide rangeof subsequently encountered factors known to cause pathologicalconditions which are associated with excessive degrees of apoptosis ofnecrosis of cells of various body organs.

Medical disorders associated with excessive degreess of apoptosis and/ornecrosis in various organs or tissues, and for which, accordingly, theprocess of the present invention is indicated for use, either as atreatment thereof or as preconditioning against the effects thereof, canbe classified into four general categories. These are:

(1) radiation exposure disorders, which include exposure to excessiveamounts of ionizing radiation such as nuclear radiation, therapeuticradiation or X-rays; or ultraviolet light (resulting in skin disorderssuch as sunburn, for example). The fact that such radiation exposuredisorders are associated with increases in apoptosis is known, forexample from Blankenberg et.al. “Dying a thousand deaths. Radionuclideimaging of apoptosis”, O. J. Nucl. Med. 1999 June; 43(2): 170-6 andvarious references cited therein; from Wong, G. H. “Protective roles ofcytokines against radiation: induction of mitochondrial MnSOD”, Biochim.Biophys. Acta 1995 May 24; 1271(1): 205-209 and various references citedtherein, from Zhao et.al. “Mitochondrial and intracellular free-calciumregulation of radiation-induced apoptosis in human leukemic cells”, IntJ Radiat Biol 1999 April; 75(4): 493-504; and from Reap E. A. et.al.,“Radiation and stress-induced apoptosis: a role for Fas/Fas ligandinteractions”, Proc Natl Acad Sci USA. 1997 May 27; 94(11):5750-5

(2) chemical exposure and ingestion disorders, which include chemicalpoisoning; food poisoning from bacterial toxins; toxic drug ingestionoverdoses and side effects; disorders from exposure to chemical warfareagents such as nerve gases and mustard gas; liver disorders fromchemicals and toxins (including alcohol); kidney disorders e.g.resulting from ingestion of aminoglycoside antibiotics, radiographiccontrast dyes or cyclosporin nephrotoxicity; hematopoietic disorders andimmunodeficiency disorders derived from drug or toxin induced bonemarrow suppression; infections from bacterial toxins; ozone exposure;solvent exposure; and the effects of immunosuppressants such ascyclosporin, cyclophosphamide or azathioprine. The fact that suchchemical ingestion and exposure disorders are associated with increasesin apoptosis is known, for example from Losser M R and Payen D,“Mechanisms of liver damage”, Semin Liver Dis, 1996 November; 16(4):357-67; from Smith K. J. et.al., Immunohistochemical studies of basementmembrane proteins and proliferation and apoptosis markers in sulfurmustard induced cutaneous lesions in weanling pigs”, J. Dermaol. Sci.1997 September; 15(3): 173-82; from Dabrowska M. I. et. al., Sulfurmustard induces apoptosis and necrosis in endothelial, cells”, ToxicolAppl Pharmacol 1966 December; 141(2>: 569-83; from Muller et.al.,“Anthracycline-derived chemotherapeutics in apoptosis and free radicalcytotoxicity (Review)”, Int J Mol Med 1998 Febuary: 1(2): 4914, andvarious references cited therein; from Healey et.al., “Apoptosis andnecrosis: mechanisms of cell death induced by cyclosporine A in a renalproximal tubular cell line”, Kidney Int, 1998 December; 54(6): 1955-66;from Hatake K et.al., “Apoptosis-gene expression in hematopoieticsystem: normal and pathologicAl conditions (Review)”, Int J Mol Med 1998January; 1(1): 121-9 and various references cited therein; from BankerD. E. et.al., “Measurement of spontaneous and therapeutic agent-inducedapoptosis with BCL-2 protein expression in acute myeloid leukemia”,Blood, 1997 Jan 1;(1):243-55; from Voetberg B. J. et.al., “Apoptosisaccompanies a change in the phenotypic . . . ”, Clin ImmunolImmunopathol 1994 May; 71(2): 190-8; and from Mountz J. D. et.al.“Autoimmune disease. A problem of defective apoptosis”, Arthritis Rheum1994 October; 37(10): 1415-20;

(3) neurological disorders such as Parkinson's disease (which involvesapoptosis of specific brain cells), senile dementia, and Alzheimer'sdisease and like diseases. The fact that such neurological disorders areassociated with increases in apoptosis is known, for example fromDesjardins P, Ledoux S “The role of apoptosis in neurodegenerativediseases,” Metab. Brain Dis. 1998 June; 13(2):79-96; from Dragunow M,McGibbon G. A. et.al. “Apoptosis, neurotrophic factors andneurodegeneration”, Rev. Neurosci. 1997 July-December;8(3-4): 223-265;from Kitamura Y, Taniguchi T, Shimohama S, “Apoptotic cell death inneurons and glial cells: implications for Alzheimer's disease”, Jpn J.Pharmacol. 1999 January; 79(1): 1-5; and from Budd S. L. and Nicholls D.G. “Mitochondria in the life and death of neurons”, Essays Biochem1998;33;43-52; and other publications both preceding and following thosedetailed above;

(4) physical trauma disorders such as physical accident injuries,wounding, thermal injuries (burns), and losses of blood such as occurduring surgery. The fact that such disorders are associated withincreases in apoptosis is known, for example from Wilson S. E,“Molecular cell biology for the refractive corneal surgeon: programmedcell death and wound healing”, J Refract Surg, 1997 March-April; 13(2):171-5.

The determination of whether or not a particular process or procedurehas an effect on apoptosis in tissues or organs of the living mammalianbody is best determined at the cellular level, e.g. by determination ofmitochondrial membrane potential or by determination of the degree ofDNA fragmentation. These measurements are described in more detail inthe specific examples which follow. A determination by such measurementsthat a process or procedure leads to a decrease in apoptosis is anindication that such a process or procedure is effective in treating orpreconditioning against any of the apoptosis related disorders listed inthe four categories above. Such a determination of apoptosis inhibitingeffects of a process or procedure, at the cellular level, in conjunctionwith a demonstrated efficacy of that process in alleviating orpreconditioning against a disorder in one of the above categories isstrong evidence of potential clinical success of that process orprocedure in alleviating or preconditioning against other disorders inthe same category.

Thus the process of the invention is primarily indicated for use bypeople who are likely to encounter conditions where they are exposed tosuch factors, such as workers in chemical manufacturing facilities,nuclear installations and the like, or physically hazardous situationssuch as emergency response teams. Potential military applicationswhereby troops may be pre-conditioned against a wide variety of hazards,will be apparent. More specific indications for use of the process arein connection with patients undergoing medical treatments, includingadministration of toxic drugs, which are accompanied by undesired sideeffects. For example, the administration of immunosuppressants such ascyclosporin, cyclophosphamide and azathioprine to assist in organtransplants and for other purposes leads commonly to apoptosis and/ornecrosis acceleration associated disorders. The use of the process ofthe present invention on patients involved in such treatments can bebeneficial, particularly since the treatment regimen for such patients,both with drugs or radiation and with the process of the invention, canbe carefully planned in advance and conducted according to a carefullycontrolled schedule.

In addition to the use of the process of the invention to pre-conditiona body or body organ against subsequently encountered factors, theprocess can also be used to control or to alleviate the symptoms of amedical disorder involving increased apoptosis and/or necrosis. The term“alleviating or protecting against the symptoms” as used herein refersto both pre-conditioning to afford protection, and treatment ofmanifested symptoms. In situations where the causative factor of themedical disorder is associated with ageing (Parkinson's disease orsenile dementia, for example), use of the process of the invention bypatients suffering from the disorder, in order to control it or toalleviate its symptoms, is the most practical use of it. Indeed,clinical tests have provided evidence of improvement in cognition andgeneral well-being of elderly patients.

Accordingly, in one aspect the present invention provides a process ofalleviating or protecting against the symptoms of a medical disorderinvolving accelerated rates of apoptosis or necrosis in a mammalianbody, said disorder being selected from radiation exposure disorders;chemical exposure and ingestion disorders; neurological disorders; andphysical trauma disorders; which comprises reducing the rate of orsusceptibility to apoptosis or necrosis of tissues and organs of themammalian body by (a) reacting an aliquot of blood from the mammalianbody ex vivo with at least one stressor selected from the groupconsisting of a temperature above or below body temperature, ultravioletlight, and an oxidative environment; and (b) administering the aliquotof blood treated in step (a) to the mammalian body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 of the accompanying drawings is a graphical presentation of theresults obtained from Example 1 described below;

FIGS. 2 and 3 of the accompanying drawings are graphical presentationsof the results obtained from Example 2 described below;

FIG. 4 of the accompanying drawings is a graphical presentation of theresults obtained according to Example 3 below.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to a preferred process of the present invention, an aliquot ofblood is extracted from a mammalian subject, preferably a human, and thealiquot of blood is treated ex vivo with certain stressors, described inmore detail below. The terms “aliquot”, “aliquot of blood” or similarterms used herein include whole blood, separated cellular fractions ofthe blood including platelets, separated non-cellular fractions of theblood including plasma, and combinations thereof. The effect of thestressors is to modify the blood, and/or the cellular or non-cellularfractions thereof, contained in the aliquot. The modified aliquot isthen re-introduced into the subject's body by any suitable method, mostpreferably intramuscular injection, but also including subcutaneousinjection, intraperitoneal injection, and oral, nasal or rectaladministration. intra-arterial injection or intravenous injection.

The stressors to which the aliquot of blood is subjected ex vivoaccording to the method of the present invention are selected fromtemperature stress (blood temperature above or below body temperature),an oxidative environment and ultraviolet light, individually or in anycombination, simultaneously or sequentially. The aliquot has a volumesufficient that, when re-introduced into the subject's body, apre-conditioning against apoptosis level is achieved in the subject.Preferably, in human patients, the volume of the aliquot is up to about400 ml, preferably from about 0.1 to about 100 ml, more preferably fromabout 5 to about 15 ml, even more preferably from about 8 to about 12ml, and most preferably about 10 ml.

It is preferred, according to the invention, to apply all three of theaforementioned stressors simultaneously to the aliquot under treatment,in order to ensure the appropriate modification to the blood. It mayalso be preferred in some embodiments of the invention to apply any twoof the above stressors, for example to apply temperature stress andoxidative stress, temperature stress and ultraviolet light, orultraviolet light and oxidative stress. Care must be taken to utilize anappropriate level of the stressors to thereby effectively modify theblood to achieve the desired effect.

The temperature stressor warms the aliquot being treated to atemperature above normal body temperature or cools the aliquot belownormal body temperature. The temperature is selected so that thetemperature stressor does not cause excessive hemolysis in the bloodcontained in the aliquot and so that, when the treated aliquot isinjected into a subject, an effective pre-conditioning against apoptosisand/or necrosis will be achieved. Preferably, the temperature stressoris applied so that the temperature of all or a part of the aliquot is upto about 55° C., and more preferably in the range of from about −5° C.to about

In some preferred embodiments of the invention, the temperature of thealiquot is raised above normal body temperature, such that the meantemperature of the aliquot does not exceed a temperature of about 55°C., more preferably from about 40° C. to about 50° C., even morepreferably from about 40° C. to about 44° C., and most preferably about42.5±1° C.

In other preferred embodiments, the aliquot is cooled below normal bodytemperature such that the mean temperature of the aliquot is within therange of from about 4° C. to about 36.5° C., even more preferably fromabout 10° C. to about 30° C., and even more preferably from about 15° C.to about 25° C.

The oxidative environment stressor can be the application to the aliquotof solid, liquid or gaseous oxidizing agents. Preferably, it involvesexposing the aliquot to a mixture of medical grade oxygen and ozone gas,most preferably by bubbling through the aliquot, at the aforementionedtemperature range, a stream of medical grade oxygen gas having ozone asa minor component therein. The ozone content of the gas stream and theflow rate of the gas stream are preferably selected such that the amountof ozone introduced to the blood aliquot, either on its own or incombination with other stressors, does not give rise to excessive levelsof cell damage. Suitably, the gas stream has an ozone content of up toabout 300 μg/ml, preferably from about 10 to about 100 μg/ml, morepreferably about 30 μg/ml, even more preferably up to about 20 μg/ml,particularly preferably from about 10 μg/ml to about 20 μg/ml, and mostpreferably about 14.5±1.0 μg/ml. The gas stream is suitably supplied tothe aliquot at a rate of up to about 2.0 litres/min, preferably up toabout 0.5 litres/min, more preferably up to about 0.4 litres/min, evenmore preferably up to about 0.33 litres/min, and most preferably about0.24±0.024 litres/min. The lower limit of the flow rate of the gasstream is preferably not lower than 0.01 litres/min, more preferably notlower than 0.1 litres/min, and even more preferably not lower than 0.2litres/min.

The ultraviolet light stressor is suitably applied by irradiating thealiquot under treatment from a source of UV light while the aliquot ismaintained at the aforementioned temperature and while the oxygen/ozonegaseous mixture is being bubbled through the aliquot. Preferred UVsources are UV lamps emitting UV-C band wavelengths, i.e. at wavelengthsshorter than about 280 nm. Ultraviolet light corresponding to standardUV-A (wavelengths from about 315 to about 400 nm) and UV-B (wavelengthsfrom about 280 to about 315) sources can also be used. For example, anappropriate dosage of such UV light, applied simultaneously with theaforementioned temperature and oxidative environment stressors, can beobtained from lamps with a power consumption of from about 15 to about30 watts and useful UV output of about 5-10 watts, arranged to surroundthe sample container holding the aliquot. Up to eight such lampssurrounding the sample bottle, operated at an intensity to deliver atotal UV light energy at 253.7 nm at the surface of the blood of fromabout 0.025 to about 10 joules/cm², preferably from about 0.1 to about3.0 joules/cm², may advantageously be used. Such a treatment provides amodified blood aliquot which is ready for injection into the subject.

The time for which the aliquot is subjected to the stressors is normallywithin the time range from about 0.5 up to about 60 minutes. The timedepends to some extent upon the chosen intensity of the UV light, thetemperature, the concentration of the oxidizing agent and the rate atwhich it is supplied to the aliquot. Some experimentation to establishoptimum times may be necessary on the part of the operator, once theother stressor levels have been set. Under most stressor conditions,preferred times will be in the approximate range of from about 2 toabout 5 minutes, more preferably about 3 minutes. The starting bloodtemperature, and the rate at which it can be warmed or cooled to apredetermined temperature, tends to vary from subject to subject.Preferably four such lamps are used.

In the practice of the preferred process of the present invention, theblood aliquot may be treated with the stressors using an apparatus ofthe type described in aforementioned U.S. Pat. No. 4,968,483 to Mueller.The aliquot is placed in a suitable, sterile, UV light-transmissivecontainer, which is fitted into the machine. The UV lamps are switchedon for a fixed period before the gas flow is applied to the aliquotproviding the oxidative stress, to allow the output of the UV lamps tostabilize. The UV lamps are typically on while the temperature of thealiquot is adjusted to the predetermined value, e.g. 42.5±1° C. Then theoxygen/ozone gas mixture, of known composition and controlled flow rate,is applied to the aliquot, for the predetermined duration of up to about60 minutes, preferably 2 to 5 minutes and most preferably about 3minutes as discussed above, so that the aliquot experiences all threestressors simultaneously. In this way, blood is appropriately modifiedaccording to the present invention to achieve the desired effects.

In operating the process of the invention, it is preferred to give apatient a course of treatments, comprising a daily or alternate daytreatment, over a period of one or two weeks. Each treatment issubstantially identical, with the same volume aliquot being extracted,stressed and re-injected. The course of treatments is scheduled to becompleted shortly before the patient is to be exposed to anapoptosis-accelerating factor as described above, for most effectivepre-conditioning against the effects thereof.

The invention is further illustrated and described with reference to thefollowing specific examples, namely animal studies conducted in approvedmanner.

The experiments reported in the examples demonstrate, by use of ananimal model system involving ischemia and subsequent reperfusion ofvarious body organs, that the process of the present invention has theeffect of reducing apoptosis and necrosis. Ischemia-reperfusion injuriesare known to involve increase of apoptosis and necrosis in the affectedorgans and tissues—see for example Saikumar p, et. al. “Mechanisms ofcell death in hypoxia/reoxygenation injury”, Oncogene 1998 Dec. 24;17(25):3341-9; and Burns A. T. et.al., “Apoptosis inischemia/reperfusion injury of human renal allografts”, Transplantation,1998 Oct. 15; 66(7): 872-6, and other publications both preceding andfollowing those. Known techniques of determination of apoptosis at thecellular level are employed in the examples. The finding that theprocess of the invention decreases apoptosis and necrosis in this modelis indicative of its utility in the various categories ofapoptosis-associated disorders discussed above.

EXAMPLE 1

Pure-bred normal beagle dogs, aged 1-2 years, equal numbers of males andfemales, were used as the experimental animals. The animals wereseparated into four groups, A, B, C and D, each group consisting of sixanimals, three males and three females. Animals of groups A and C weresubjected to the process of the invention, by being subjected to two10-day courses of daily removal of an 8 ml aliquot of blood,extracorporeal treatment of the aliquot with oxygen/ozone, UV radiationand heat, and re-administration of 5 ml of the treated aliquot to thesame animal, by intramuscular injection.

Each such treatment was conducted as follows.

An 8-ml aliquot of blood was extracted from the animal, treated withsodium citrate (2 ml) and placed in a sterile container. It wassubjected simultaneously to the UV radiation, oxygen/ozone gas oxidativeenvironment and elevated temperature stressors, in an apparatus asgenerally described in the aforementioned Mueller U.S. Pat. No.4,969,483. More specifically, the blood sample in the sterile,UV-transparent container was heated using infra-red lamps to 42.5° C.,and whilst being maintained at that temperature, it was subjected to UVradiation of wavelength 253.7 nm under the preferred conditionspreviously described. Simultaneously, a mixture of medical grade oxygenand ozone, of ozone content 13.5-15.5 ug/ml was bubbled through theblood sample at a flow rate within the range from 60-240 mls/min. Thetime of simultaneous UV exposure and gas mixture feed was 3 minutes. A 5ml portion of the treated blood aliquot was reinjected intramuscularlyinto each test animal.

Each animal of groups A and C, receiving the courses of treatmentaccording to the invention, experienced a three week rest period betweenthe 10-day courses of treatment. Groups B and D were the control groups,given two 10-day courses of daily injections of 5 ml of physiologicalsaline, with a three-week rest period between the 10-day courses.

One day following the second course of injections, the animals wereanaesthetized under light gas anaesthesia, and the right kidney of eachanimal was removed through a back incision. An occlusive clip was placedon the remaining renal artery and vein, to expose the left kidney totransient ischemia, for 60 minutes; Then the clip was removed to allowreperfusion of the kidney by normal blood flow.

The animals were observed for 6 days after the ischemia procedure, andthen sacrificed. The ischemic kidney of each animal was surgicallyremoved and divided into two parts. One part was kept frozen at −80° C.,and the other part was fixed in 10% formalin for immuno- and routinehistopathology studies.

Mitochondrial membrane potential was measured in proximal tubular cellsisolated from the ischemic and control kidneys, both at the time ofremoval of the control kidney and following sacrifice. For this purpose,dog kidney proximal tubes were purified from normal or ischemic kidneycortexes by the collagenase treatment procedure described by Marshanskyet. al., “Isolation of heavy endosomes from dog proximal tubes insuspension”, J. Membr. Biol 153(1), 59-73, 1996. Renal mitochondria wereisolated in suspension by differential centrifugation (see Marshansky,“Organic hydroperoxides at high concentrations cause energization andactivation of AATP synthesis in mitochondria”, J. Biol. Chem. 264(7),3670-3673. 1989. after tissue homogenization in a buffer containing 250mM sucrose, 10 mM HEPES-Tris (pH 7.5), and 250 μM EDTA. Cell debris wasremoved by centrifugation at 10,000 g for 30 minutes. The mitochondriawere washed with the sucrose/HEPES buffer without EDTA.

Mitochondrial membrane potential was measured as described by Kroemer,G., Zamzam, N. and Susin, S. A., “Mitochondrial control of apoptosis”,(Review) Immunology Today (1997) v.18, p 44-51; with JC-1 dye—seeSalvioli et.al., “JC-1, but not DiOC6(3) or rhodamine 123, is a reliablefluorescent probe to assess delta psi changes in intact cells:implications for studies on mitochondrial functionality duringapoptosis”, FEBS Letters 411 (1), 77-82. 1987. JC-1 fluorescence in thesuspension of purified mitochondria from normal and ischemic kidneys wasmonitored continuously on a Deltascan Model RFM-2001 spectrofluorimeter(Photon Technology International, South Brunswick, N.J.). The excitationwavelength was 490 nm (slit width 2 nm) and the emission wavelength was590 nm (slit width 4 nm). The signals were recorded using Felix®(Version 1.1) software. All measurements were performed with continuousstirring at 37° C. The incubation buffer for measurement ofmitochondrial membrane potential contained 200 mM sucrose, 5 mM MgCl₂, 5mM KH₂PO₄, 0.1 μM of JC-1 and 30 Mm HEPES-Tris (pH 7.5). Theconcentrations of the substrate and inhibitors were 10 mM succinate, 0.1μM rotenone with or without 0.1 μM FCCP. Proximal tubule mitochondrialmembrane potential was estimated in the right (control) kidney prior toischemia and in the left (ischemic) kidney after sacrifice of the dogson day 6 following ischemia and was estimated as difference of JC-1fluorescence after uncoupling of mitochondria with FCCP as shown in theaccompanying FIG. 1A. For each measurement, 50 μg protein of purifiedmaterial was used.

JC-1 fluorescence is proportional to the mitochondrial membranepotential. The contralateral nephrectomized kidney served as control. Asis clear from the FIG. 1B, the treatment process of the invention didnot modify the membrane potential of the non-ischemic control rightkidney (p=0.445 for treated vs saline). However, the ischemic kidney ofthe saline-injected animals showed significantly lower (p<0.05)fluorescence compared to the control kidney. The stress treatmentaccording to the invention prevented the uncoupling of mitochondriaduring ischemic/reperfusion, and membrane potential showed nosignificant difference (p=0.244) between ischemic and control kidneys.This parameter remained significantly higher (p=0.0006) vssaline-injected dogs) in the ischemic kidneys of dogs pretreatedaccording to the process of the invention for at least 6 dayspost-reperfusion.

These results indicate that the process of the invention effectsprotection of the kidney against apoptosis and/or accelerates recoveryat the mitochondrial level. Accordingly the process of the invention isindicated for preconditioning of the cells, tissues and organs of amammalian body against subsequently encountered factors which willnormally accelerate apoptosis.

Specifically, the preservation of mitochondrial membrane potentialevidences the capacity of the therapy to protect mitochondria, andthereby to precondition cells against apoptosis.

EXAMPLE 2

A group of 12 male SHR rats was treated with either injections of pooledblood stressed as described in Example 1 above, or, in control animals,with injections of saline. Since the blood from all of the animals ofthis genetic strain is identical, blood from one animal of this samestrain was treated by the process of the invention for administration tothe test animal. The blood was treated with sodium citrate asanti-coagulant, and placed in a sterile container They received eitherinjections of 150 μl of stressed blood on days-14 and -13 followed by arest period of 11 days and a third injection the day before ischemicsurgery, or injections in parallel with saline. On the day of surgery,the rats were anaesthetized with light flurane, and the right kidney wasremoved through a mid-abdominal incision. The left kidney was thensubjected to transient ischemia by occlusion of the left renal arteryand vein using a micro-clip. The skin was then temporarily closed. After60 minutes of occlusion, the clip was removed and the wound was closedwith a suture. The animals were sacrificed 12 hours after reperfusion.

The ischemic and non-ischemic kidneys of the test animals were removedand subjected to DNA laddering tests. Oligonucleosomal DNA fragmentationinto 180 to 200 base pairs is a specific pattern which appears as aladder after agarose gel electrophoresis in various organs undergoingapoptosis. To estimate the degree of DNA fragmentation in the kidneycortex, an aliquot of pulverized kidney cortex was weighed and totaltissue DNA was extracted by the phenol-chloroform procedure after tissuedigestion with proteinase K and RnaseA in the presence of EDTA. One μgof extracted DNA was labeled by enzymatic assay using terminaldeoxynucleotidyl transferase with P³²-dCTP (see Teiger et.al.,‘Apoptosis in pressure overload-induced heart hypertrophy in the rat’,J. Clin. Invest. 97, 2891-2897, 1996). Increasing quantities ofradio-labelled DNA were loaded onto 1.5% agarose gels. Afterelectrophoresis, DNA was transferred onto nylon membranes (Hybond) andthe radioactivity associated with 150 to 1500 bp DNA fragments wasquantified in a PhosphorImager (Molecular Dynamics). A regression linefor each sample was drawn for the radioactivity as a function of DNAloaded on the gel (see deBlois et.al., ‘Smooth muscle cell apoptosisduring vascular regression in spontaneously hypertensive rats.’Hypertension 29, 340-349, 1997). The slope of the linear regression lineserved as a DNA fragmentation index (cpm/pixel per μg DNA).

The results from ischemic-reperfused (I/R) kidneys and from normal,non-I/R kidneys, all from animals which did not receive injections ofstressed blood, are shown graphically on FIG. 2, a plot of the slope ofthe regression lines for the various samples (vertical axis) againsttime after initiation of reperfusion. The DNA laddering, indicative ofDNA fragmentation, was clearly increased in the ischemic kidney cortexcompared to the contralateral non-ischemic organ and the maximalattained at twelve hours returned to near basal values by 48 hours.Twelve hours was thus selected as the time point for study of the effectof the stressed blood of the invention on early ischemia-induced renalapoptosis.

FIG. 3A of the accompanying drawings is a picture of the electophoresisgel of the fragmented DNA, in the 150-1500 bp range, radio-labeled asdescribed to attach radioactivity labels to the DNA fragments. Trace Sderives from DNA of kidneys from animals which received salineinjections prior to kidney ischemia-reperfusion, and trace V derivesfrom DNA of kidneys of animals which received injections of the stressedblood prior to kidney ischemia-reperfusion. The Figure shows that 60minutes renal ischemia induced a clear accumulation of fragmented DNA inboth groups of rats at 12 h but the level of this parameter wassignificantly lower (p<0.05) in animals receiving the treated blood.FIG. 3B quantifies the amount of irradiation from the samples, inarbitrary units, and shows that DNA fragmentation-laddering occurs inboth S and V samples as a result of ischemia/reperfusion, but that theextent is markedly reduced in V samples as compared with S samples. Theresults presented on FIG. 3B are the means of six animals in each case.

These results confirm that the cytoprotective effect of theadministration of stressed blood according to the invention on renalreperfusion injury involves the inhibition of early or late apoptosis.

EXAMPLE 3 Heart Studies—Protection of Removed Organs

Experiments were carried out in rats, more specifically in maleSprague-Dawley rats to demonstrate the protection of removed organs,deprived of the donor's blood, against a sustained ischemic insult astypically observed with classical ischemic preconditioning protocol (K.Przylenk and R. A. Kloner, Progress in Cardiovasc. Dis. vol 40: 517-547,1998).

Two groups of four rats, 270-285 g body weight, were used. One group ofrats (n=4) received a saline injection and served as controls. The othergroup of rats received blood treated by a protocol in accordance withthe invention. Since the blood from all of the animals of this geneticstrain is identical, blood from one animal of this same strain wastreated by the process of the invention for administration to the testanimal. The blood was treated with sodium citrate as anticoagulant, andplaced in a sterile container. It was heated and subjectedsimultaneously to the UV stressor and oxygen/ozone stressor in theamounts and under the conditions set out in Example 1 above.

Each test animal received on day 1 an injection of 150 μl of the treatedblood, followed by a 10-day rest period. Then each animal received a 150μl injection of treated blood on both day 12 and on day 13. Each controlanimal received similar injections, on the same schedule, ofphysiological saline. The animals were then sacrificed on day 14.

From each animal, the heart was removed and perfused ex vivo accordingto the Langendorf mode with non-recirculating Krebs Henseleit buffergassed with 95% oxygen/5% carbon dioxide pH 7.4, containing glucose asenergy substrate. The heart was submitted to an ischemia-reperfusioninsult as typically is used in studies of cardiac ischemicpreconditioning (see for example R. T. Rowland et.al., Am. J. Physiol.272, H2708-H2715; E. O. Weselcouch et.al., Cardiovasc. Res. 29; 126-132,197). Briefly, after a 20 minute equilibration period under normoxia,the heart was submitted to a 25 minute global ischemia at 37° C. Then,it was reperfused for 45 minutes as follows: (i) for the initial 25 minof reperfusion, the heart was allowed to beat spontaneously, the (ii) itwas paced using pacing wires fixed to the right atrium to achieve arhythm of 300 beats/min.

FIG. 4 indicates data measuring the perfusion protocol for the lactatedehydrogenase released into the effluent perfusate, an index of cellularnecrosis, as evaluated by a standard enzymatic assay. In FIG. 4 of thedrawings, the curve based on the triangular-form points is derived fromorgans of animals which received blood treated with stressors asdescribed. The curve based on square-form points is derived from theorgans of the control animals which received saline solution. FIG. 4indicates a significant reduction in LDH release (cumulative LDH releaseduring the 45 minute reperfusion period; p<0.05; treated vs saline),indicative of significantly reduced cell necrosis in organs treated withstressor as described.

1. Use in alleviating or protecting against the symptoms of a medicaldisorder involving accelerated rates of apoptosis or necrosis in amammalian body, said disorder being selected from radiation exposuredisorders; chemical exposure and ingestion disorders; neurologicaldisorders; and physical trauma disorders; through reducing the rate ofor susceptibility to apoptosis or necrosis of tissues and organs of themammalian body, of an aliquot of blood from the mammalian body which hasbeen extracted therefrom and reacted ex vivo with at least one stressorselected from the group consisting of a temperature above or below bodytemperature, ultraviolet light, and an oxidative environment.
 2. Useaccording to claim 1 wherein the aliquot of blood which is ex vivoreacted has a volume from about 0.1-100 ml.
 3. Use according to claim 1or claim 2 wherein said at least one stressor is a temperature in therange from about −5-55 C.
 4. Use according to claim 3 wherein said atleast one stressor is a temperature in the range from about 40-50 C. 5.Use according to claim 1 or claim 2 wherein said at least one stressoris an oxidative environment comprising a mixture of ozone and medicalgrade oxygen, bubbled through the blood aliquot.
 6. Use according toclaim 5 wherein the gaseous mixture has an ozone content of from about10-100 pg per ml.
 7. Use according to claim 1 or claim 2 wherein said atleast one stressor is ultraviolet light in the UV-C band wavelength. 8.Use according to any preceding claim wherein all three said stressorsare applied to the aliquot simultaneously.
 9. Use according to claim 8wherein said stressors are applied for a period of time from 0.5 to 60minutes.
 10. Use according to claim 9 wherein the time is from about 2-5minutes.
 11. Use according to any preceding claim wherein the medicaldisorder is a radiation exposure disorder.
 12. Use according to any ofclaims 1 to 10 wherein the disorder is an ionizing radiation exposuredisorder or an ultraviolet light exposure skin disorder.
 13. Useaccording to any of claims 1 to 10 wherein the medical disorder is achemical exposure or chemical ingestion disorder.
 14. Use according toclaim 13 wherein the disorder is chemical poisoning; food poisoning frombacterial toxins; toxic drug ingestion overdoses and side effects;disorders from exposure to nerve gases and mustard gas; liver disordersfrom chemicals and toxins; kidney disorders resulting from ingestion ofaminoglycoside antibiotics, radiographic contrast dyes or cyclosporinnephrotoxicity; hematopoietic disorders and immunodeficiency disordersderived from drug or toxin induced bone marrow suppression; infectionsfrom bacterial toxins; ozone exposure; solvent exposure; or the effectsof immunosuppressants.
 15. Use according to any of claims 1 to 10wherein the medical disorder is a neurological disorder. 16-18.(canceled)